Different types of bone grafts have been used more or less successfully to replace lost bone tissue and improve healing of bone defects for the purpose of restoring the function with and without fixed implants. In autogenous transplantation, usually from the patient's own iliac crest, it is among other things the amount of bone and the degree of resorption that affect the result of the treatment. Autologous bone grafting usually requires more than one operation to achieve a satisfactory result and causes considerable postoperative pain to the patient. In homologous bone grafting, use is made of, for instance, demineralised bone matrix from a so-called bone bank. Tissues and structure which have been lost owing to diseases or injuries can today to some extent be replaced by prosthetic constructions which are mechanically fixed to the skeleton. Artificial hips, artificial knee-joints and dental implants are examples of how lost tissue, structure and function can be replaced by this type of construction.
Replacing lost teeth by placing dental implants in the skeleton (jaws) has a high frequency of success provided that there is a sufficient amount of bone of good quality in the close vicinity of and round the implant. After an individually adapted time of healing (3 months–2 years), prosthetic constructions can in most cases be secured to the osseointegrated implants. Some patients have, owing to many years of lack of teeth, obtained impaired conditions than others for obtaining treatment by means of bone-secured implants. It is mainly areas in the upper jaw that have suffered most from bone destruction when the teeth are lost owing to anatomical conditions, but also areas far back in the lower jaw can have a poor quality for implant treatment.
General states of ill-health such as osteoporosis and local defects and lack of bone owing to, for instance, acute injuries, congential defects, chronical infections or local biological processes such as cysts and tumours in the jaws in most cases affect and may even make treatment with implants impossible if bone is not added or replaced in some way to increase the amount of bone locally round the implants and, thus, the initial stability.
For patients who have been treated with implants and bone replacement materials, it is important to reduce the time of osseointegration and guarantee a higher frequency of success after installation than is possible today. For decades, experiments have been made to replace bone with organic and synthetic materials from different sources, see review, Smiler et al (1992)1 including resorbable and non-resorbable polymers, bioactive glass, calcium phosphate compounds, calcium carbonates and naturally occurring materials such as cow bone and coral. Transplanting bone from the patient, so-called autograft, is as described above an alternative, but a relatively extensive operation which requires specialist competence, hospital treatment, an extended healing period for the transplant of at least six months and additional inconvenience and pain to the patient. Demineralised freeze-dried bone from a so-called bone bank and spongious bone with mineral from cow bone also result in an extended period of healing, a risk of immunological reactions and infectious states, but also a risk in other aspects including the frequency of success.
There is thus a great need, both with specialists and non-specialists, for being able to apply an easily accessible safe preparation for restoring bone in connection with implant treatment of patients having an insufficient volume of bone and/or too poor bone quality.
An object of the invention is to provide a preparation for restoring bone, which enables implant treatment for patients in various situations especially in areas that otherwise cannot be treated and/or have a poorer prognosis. Calcium phosphate compounds are so-called biocompatible materials, i.e. materials which are mildly reactive with the environment, i.e. promote repair and integration of, for instance, an implant, see Jarcho2 and Lemons3. The commonest form of calcium phosphate compound that is used to stimulate ossification is hydroxylapatite (HA), Ca10(PO4)6(OH)2, but also other compounds containing calcium and phosphate ions exist and resemble the inorganic ingredients in skeleton and enamel, see Daculsi et al (1997)4. Stoichiometric HA, Ca10(PO4)6(OH)2, with a Ca:P ratio of 1.67 is seldom found in vivo. Calcium is to some extent replaced by other ions, such as magnesium, sodium, aluminium, strontium, carbonate, fluorine and chlorine, depending on, inter alia, age, food, sex etc. of the individual. HA may be present in a ceramic and in a non-ceramic form where the degree of crystallinity may vary depending on the temperature at which the calcium phosphate compound is prepared, Ricci et al (1992)5.
Calcium phosphate compounds such as hydroxylapatite are commercially available and produced by many companies; Impladent Ltd, Holliswood, N.Y., USA, Asahi Optical Co, Ltd, Tokyo, Japan, Interpore Int. and Irvine, Calif., USA. The materials are produced with different properties such as the size of the granules/particles, the degree of resorbability and the chemical composition.
The particles/granules being resorbed do so slowly after application in the bone tissue. It is considered that from the beginning the granules physically take up room in the defect and thus allow an accelerated integrating process compared with an empty defect. While the new bone is forming, mineralising and remodelling, the granules are resorbed slowly for about 3–8 months depending on patient-related factors including the size of the defect and the age of the patient.
There are experimental studies, Hürzeler et al (1995)6 and Wetzel et al (1995)7 and clinical studies, Smiler et al (1992)1, Ricci et al (1992)5, Judy (1986)8, Wagner (1989)9, and Corsair (1990)10, which demonstrate a certain effect of resorbable granules (Osteogen®) mixed with, for instance, patient blood, common salt or above all in combination with demineralised freeze-dried bone. The possibility of making a filling in sinus maxillaris (sinus of the upper jaw) to increase the possibilities of implant fixing in dogs shows that resorb able HA granules function well and the product is suitable for use to stimulate bone formation round dental implants, Wetzel et al (1995)7. However, the granules are mixed with patient blood or common salt, which makes the product difficult to handle. A great drawback of this form/technique of preparation is that it is technically difficult to pass the mass of granules to the defect/cavity area. Having applied the granules, there is a great risk that blood and other body fluids from the area of the wound dilute and transport the material away from the area of application. A further drawback of uncontrolled mixing of, for instance, common salt or the patient's blood is that the risk of contamination of the preparation increases.
Alpher et al (1989)11 have tried to solve the handling problems. However this reference shows that a mouldable hydroxylapatite preparation based on phospholipids and stearic acid generally does not stimulate the formation of new bone or bone growth, but instead the results indicate a reduction of the bone formation.
Lipids can be divided into different classes. Triglycerides are the most frequent class of lipids and are an important depot of energy in cells. Triglycerides are either built up or decomposed in the body by the intermediary of diglycerides from or into monoglycerides and fatty acids. The body also contains different types of membrane lipids, for example phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin, cholesterol, mono- and digalactosyldiacylglycerol.
Phospholipids can be prepared fully synthetically but also be cleaned of biological raw materials such as plants or animals. Examples of raw materials are egg yolk, vegetable oils such as soybean oil, rapeseed oil.
It is also preferred for the preparation to contain anti-oxidants selected according to known principles or naturally occurring. An example of an advantageous antioxidant in this case can be tocopherol. Liposomes consist of a spherical shell of amphiphilic lipids containing an aqueous phase. The potential of the lipid vesicles as carrier of drugs has been studied and described in a number of articles. Huang et al12 have suggested that negatively charged liposomes can affect the mineralising processes of newly formed bone. This concept was tested in a defect model in miniature swine, however, without any effect. Bone formation between uncoated and liposome-coated calcium phosphate compounds was compared and the uncoated calcium phosphate compounds were surrounded by more bone and riper bone than the liposome-coated ones. In an experimental study by Raggio et al (1986)13, the authors show that complex acid lipids affect the precipitation of hydroxylapatite mineral in a physiological environment.
Recently some research has focused on the application of introducing exogenic molecules into cells by means of lipid complexes. These new lipids have an important clinical application for drug delivery and gene therapy. Since the lipids can be tailored to have different physical properties, the application may vary, Ashley et al 1996)14 and Barber et al (1996)15.
Different systems based on calcium phosphate granules and lipid carriers are described in literature, see for instance EP 0 429 419 which discloses a system where calcium phosphate, especially hydroxylapatite, is used as bone substitute material. In this case, use is made of a monoglyceride-based carrier, which may cause a drawback in implant treatment since preliminary studies indicate that encapsulation may occur, which in turn can have a negative effect on implant integration.
Various systems for the release of pharmaceutical preparations containing bioceramic granules and lipid have also been described in literature, see for instance JP 2,631,890. As examples of different carriers for drugs and molecules that are to be released in bone tissue, mention can be made of collagen, lipids, polymers (for instance PLA/PGA and hyaluronic acid) and ceramics.
A large number of studies demonstrate that mineral deposition in cartilage that is being calcified is only found in vesicles containing phosphatidyl serine and alkaline phosphatase, and that the endocondral calcification process in the growth plate in the epiphysis can be mediated by these. Matrix vesicles and the negatively charged phospholipids therein seem to be involved in the initial formation of calcium hydroxylapatite crystals by way of the interaction between calcium and phosphate ions with phosphatidyl serine in the formation of phospholipid:calcium:phosphate ion complex, Boyan et al (1989)16.
It is mainly the systemically blood calcium controlling hormones which in different ways control bone cells and, thus, keep the bone mass of the body in equilibrium. In recent years, many studies have been made which indicate that certain biopolymers of the polypeptide type produced by bone cells themselves and/or blood cells from bone marrow or in inflammation after, for instance, trauma, have an important and probably a more immediate importance for activating the individual cells in connection with the bone formation process.
Bone formation and bone resorption are connected to each other. Systemically and locally produced factors control the processes. Many of the growth factors may have different effect on different cells. For instance, PTH and vitamin D can stimulate bone resorption and remodelling by means of the bone-forming cells, cf. Nijweide et al (1986)17. On the other hand, the bone-forming cells can be stimulated by TGF beta released of matrix during the bone resorption process, cf. Pfeilschifter et al (1990)18.
The growth factors and cytokines that are produced by bone cells may have an autocrine or paracrine effect. Examples hereof are: TGF, IGF-I and IFG-II, Beta2 Microglobulin, PDGF and CSFs. Thrombocyte-derivated factors such as TGF, PDGF and EGF, but also interleukins, TNFs and Interferon gamma are factors of hematologic origin which have effect on the bone-forming cells. Growth factors which are stored in the bone matrix are the largest reservoir for growth factor activity. The factors stored are, as mentioned above, TGF, IGF-I and IGF-II, Beta2 Microglobulin, PDGF, but also FGF. Bone morphogenetic proteins, BMP, and osteogenine belong to the TGF family. BMP is usually combined with decalcified bone matrix and collagen, cf. Sampath and Reddi (1981)19 and Saito et al (1994)20. Kuboki et al (1995)21 have proved that BMP induces only bone better if the HA carrier consists of particles which are porous compared with non-porous.
In a newly published experimental study by Urist et al (1997)22, different systems for administration of the growth factor BMP-2 and its effect on bone formation were investigated. The authors suggest that lipids extracted from bone can function well as a carrier of bone-stimulating peptides in the bone formation process.
Other molecules or ions which can bind strongly to crystal surfaces are, for example, bisphosphonates which can affect osteoblasts and thus dissolution of calcium phosphate compounds in the skeleton, cf. Ebrahimpour et al (1995) 23.
According to the invention, the preparation for restoring bone is a mixture of resorbable calcium phosphate granules and/or a carrier of a biopolymer or lipid type, where the lipid contains an esterified fatty acid selected from the group consisting of triglycerides, diglycerides or phospholipids or combinations thereof. The invention aims at overcoming the difficulties described above and constituting a preparation which easily and in a repetitive manner can be used in connection with bone implants. More specifically, the inventive material is intended to withstand dilution and transporting away from the area of application. Such a mixture can be given the “correct” consistency depending on the type of application, it can be made, for example, mouldable, and it is easy to handle, control and apply.
A desirable weight ratio between calcium phosphate and phospholipid is in the order of 70:15 to 60:40. A desirable weight ratio between phospholipid and water or other water-based liquids is in the order of 1:2 to 10:1, preferably 3:2 to 4:1.
With reference to the water-based liquid that is used to make the preparation mouldable, pure water, an isotonic saline solution or a pharmaceutically acceptable solution are preferred. In some cases when the preparation is being produced in situ, body fluids including blood can be used.
As mentioned by way of introduction, the preparation for restoring bone consists of a mixture of resorbable calcium phosphate granules and a carrier of a biopolymer or lipid type. To be applied in connection with a bone implant and be kept in the area of application, it is important for the mixture to be mouldable and to have the correct consistency. If the particles are transported away from the area of application, they could cause irritation or complications in other positions in the body.
The calcium phosphate granules should have a Ca/P ratio of between 1 and 2. The granules should have an average diameter of 0.05–5 mm and a micro/macro porosity of 0–80%.
In a study made by Neo et al (1992)24, the interface between bioactive ceramics and bone was studied by using scanning and transmission electron microscopy. Calcium phosphate granules having an average diameter of 0.1 to 0.3 mm were studied and characterised in respect of resorbability. After 8 weeks, the non-resorbable granules were connected with bone by a thin Ca-P-rich layer consisting of fine apatite crystals, however, different from those in bone in respect of shape, size and orientation. The resorbable granules, however, had direct contact with the bone. The surface of the granules was coarser owing to degradation, and analyses demonstrated that bone grew into the smallest surface irregularities. In another study made by Kitsugi et al25 four types of calcium phosphate ceramics were compared. The Ca/P ratio was in this case 1, 1.5 and 1.66 and the size of the particles (granules) varied between 0.15 and 0.3 mm. Observations made by transmission electron microscopy showed that the Ca/P ratio did not affect the connection and contact between the particles and the surrounding bone.
According to the invention it is necessary to have a carrier for the resorbable calcium phosphate granules, which may consist of a biopolymer or a lipid containing esters of fatty acids, such a triglyceride, diglyceride, or phospholipids or combinations thereof, for instance some of the lipids described in WO 95/34287.
Preferably, the calcium phosphate granules are distributed in a lamellar, liquid crystalline phase which contains at least one phospholipid and forms either in the body or earlier.